9 Геном, плазмиды, вирусы (Лекции), страница 7

The genes for these polypeptides are divided into segments, and clusters containing multiple versions of each segment existin the genome. One version of each segment is joined to create a complete gene.The organization of the DNA encoding the kappa light chains ofhuman IgG and the process by which a mature kappa light chain isgenerated are shown in Figure 24-38b. In undifferentiated cells, thecoding information for this polypeptide chain is separated into threesegments. The V (variable) segment encodes the first 95 amino acidresidues of the variable region, the J (joining) segment encodes theremaining 12 amino acid residues of the variable region, and the Csegment encodes the constant region.

There are about 300 different Vsegments, 4 different J segments, and 1 C segment. As a stem cell inthe bone marrow differentiates to form a mature B lymphocyte, one Vand one J are brought together by site-specific recombination. This iseffectively a programmed DNA deletion event, and the interveningDNA is discarded.

There are 300 x 4 = 1,200 possible combinations.The recombination process is not as precise as the site-specific recombination described earlier, and some additional variation occurs in thesequence at the V - J junction that adds a factor of at least 2.5 to thetotal variation possible, so that about 2.5 x 1,200 = 3,000 differentV - J combinations can be generated. The final joining of this V-J combination to the C region is accomplished by an RNA-splicing reactionafter transcription (Fig.

24-38b). RNA splicing will be described in thenext chapter. The genes for the heavy chains and lambda light chainsare formed similarly. For heavy chains, there are more gene segmentsand more than 5,000 possible combinations. Because any heavy chaincan combine with any light chain to generate an immunoglobulin,there are at least 3,000 x 5,000 or 1.5 x 107 possible IgGs. Additionaldiversity is generated because the V sequences are subject to high mutation rates (of unknown mechanism) during B-lymphocyte differentiation.

Each mature B lymphocyte produces only one type of antibody,but the range of antibodies produced by different cells is clearly enormous. The enzymes that catalyze these gene rearrangements have notbeen isolated, but sequences critical to the V - J joining process that arepresumably recognized by these enzymes have been identified.This recombination process helps to illustrate the principle thatrecombination does not destroy the integrity of the genetic materialthat the replication and repair processes attempt to maintain.

Here wesee a precisely orchestrated process that occurs only in specialized cells(germ-line DNA is not affected) and enables the organism to makemuch more efficient use of its genetic information resource.Transposable Genetic Elements Move fromOne Location to AnotherFinally, we consider the recombination of transposable elements ortransposons. Transposons are segments of DNA, found in virtually866Part IV Information PathwaysRNA ProcessingMany of the RNA molecules in bacteria and virtually all of the RNAmolecules in eukaryotes are processed to some degree after they aresynthesized. Many of the most interesting molecular events in RNAmetabolism are to be found among these postsynthetic reactions. Thestudy of these processes has revealed that some of them are catalyzedby enzymes made up of RNA rather than protein.

The discovery ofcatalytic RNAs has brought on a revolution in thinking about RNAfunction and about the origin of life.A newly synthesized RNA molecule is called a primary transcript. Perhaps the most extensive processing of primary transcriptsoccurs in eukaryotic mRNAs and in tRNAs of both bacteria and eukaryotes. A primary transcript for a eukaryotic mRNA typically contains sequences encompassing one gene. The sequences encoding thepolypeptide, however, usually are not contiguous.

Instead, in the majority of cases, the coding sequence is interrupted by noncoding tractscalled introns; the coding segments are called exons (see the discussionof introns and exons in DNA, p. 798). In a process called splicing, theintrons are removed from the primary transcript and the exons joinedto form a contiguous sequence specifying a functional polypeptide.Eukaryotic mRNAs are also modified at each end. A structure called acap is added at the 5' end, and a polymer containing 20 to 250 adenylate residues, poly(A), is added to the 3' end.

These processes are outlined in Figure 25-10 and described in more detail below.The primary transcripts of most tRNAs (in all organisms) are alsoprocessed by the removal of sequences from each end (called cleavage)and sometimes by the removal of introns (splicing).

Many bases intRNAs are also modified; mature tRNAs are replete with unusualbases not found in other nucleic acids.The ultimate postsynthetic modification reaction is the completedegradation of the RNA. All RNAs eventually meet this fate and arereplaced with newly synthesized RNAs.

The rate of turnover of RNAsis critical to determining their steady-state level and the rate at whichcells can shut down expression of a gene whose product is no longerneeded.Transcriptionand 5' cappingDNAFigure 25-10 Formation of the primary transcriptand its processing during maturation of the mRNAin a eukaryotic cell. The 5' cap (in red) is addedbefore synthesis of the primary transcript is complete. Noncoding sequences following the last exonare shown in orange.

Splicing may occur either before or after the cleavage and polyadenylationsteps. All of the processes represented here takeplace within the nucleus."'^ _ _._.~--xIntronExonv...RNA polymeraseCompletion ofprimary transcriptPrimarytranscript>f5'Cleavage,polyadenylation,and splicing IMaturemRNA5 -1Chapter 25 RNA Metabolism867The Introns Transcribed into RNA Are Removed by SplicingIn bacteria, a polypeptide chain is generally encoded by a DNA sequence that is colinear with the amino acid sequence, continuing alongthe DNA template without interruption until the information neededto specify the polypeptide is complete. The notion that all genes arecontinuous was unexpectedly disproven in 1977 with the discovery thatthe genes for polypeptides in eukaryotes are often interrupted by thenoncoding sequences now called introns.

Introns are present in thevast majority of genes in vertebrates; among the few exceptions are thegenes that encode certain histones. The occurrence of introns in othereukaryotes is variable. Most genes in the yeast Saccharomyces cerevisiae lack introns, although introns are more prevalent in the genes ofsome other yeast species.

Introns are also found in a few prokaryoticgenes.Introns are spliced from the primary transcript, and exons arejoined to form a mature, functional RNA. Introns were discoveredwhen mRNA and the DNA from which it was derived were comparedusing methods such as that illustrated in Figure 25-11. If the DNAcontaining a gene is completely denatured and then renatured in thepresence of the mature RNA derived from the gene, an RNA-DNAhybrid is formed. This kind of experiment revealed DNA sequencesthat were not present in the RNA and therefore were looped out as inFigure 25-11.

Experiments using this and other methods have shownthe presence of multiple introns in many genes, with some genes interrupted by introns more than 40 times. In eukaryotic mRNAs mostexons are less than 1,000 nucleotides long, with many clustered in the100 to 200 nucleotide size range. Most exons therefore encode polypeptide chains that are 30 to 50 amino acids long. Introns are much morevariable in size (50 to 20,000 nucleotides).

Genes of higher eukaryotes,including humans, typically have much more DNA devoted to intronsthan to exons; it is not uncommon to find genes that are 50,000 to200,000 nucleotides long and that contain numerous introns.There are four classes of introns. The first two, called group I andgroup II, share some key characteristics but differ in the details oftheir splicing mechanisms. Group I introns are found in some nuclear,mitochondrial, and chloroplast genes coding for rRNAs; group IIintrons are generally found in the primary transcripts of mitochondrialor chloroplast mRNAs. Both groups share the property that no highenergy cofactors (such as ATP) are required for splicing.

Both splicing(a)AAA(A)^ (3'(b)(c)Figure 25-11 Defining the structure of the chickenovalbumin gene by hybridization. Mature mRNAwas hybridized to denatured DNA containing theovalbumin gene, and the resulting molecules werevisualized with the electron microscope. Some regions of the DNA have no complement in themRNA because of splicing of the primary transcript. The resulting single-stranded DNA loops areevident in the electron micrograph (a). The loopsdefine the locations and sizes of introns.

Theintrons are labeled A to G and the seven exonsare numbered in the interpretive drawing (b). Thepoly(A) tail defines the 3' end of the mRNA. The Lsequence encodes a signal sequence that targets theprotein for export from the cell, (c) A linear representation of the ovalbumin gene showing intronsand exons.868Part IV Information PathwaysGuanosinemechanisms involve two transesteriflcation reaction steps (Fig.25-12). A 2'- or 3'-hydroxyl group of a ribose makes a nucleophilicattack on a phosphorus, and in each step a new phosphodiester bond isformed at the expense of the old, maintaining an energy balance.

Notethat these reactions are very similar to the DNA breaking and rejoining reactions promoted by topoisomerases (Chapter 23) and sitespecific recombinases (Chapter 24).The group I splicing reaction requires a guanine nucleoside or nucleotide cofactor. This cofactor is not used as a source of energy; instead, the 3'-hydroxyl group of guanosine is used as a nucleophile inthe first step of the splicing pathway. The guanosine 3'-hydroxyl formsa normal 3',5'-phosphodiester bond with the 5' end of the intron (Fig.25-13). The 3'-hydroxyl of the exon that is displaced in this step thenacts as a nucleophile in a similar reaction at the 3' end of the intron.The result is precise excision of the intron and ligation of the exons.In group II introns the pattern is similar except for the nucleophile in the first step. Instead of an external cofactor, the nucleophileis the 2'-hydroxyl group of an adenylate residue within the intron(Fig.